Indirect Mechanisms of Transcription Factor‐Mediated Gene Regulation during Cell Fate Changes

Abstract Transcription factors (TFs) are the master regulators of cellular identity, capable of driving cell fate transitions including differentiations, reprogramming, and transdifferentiations. Pioneer TFs recognize partial motifs exposed on nucleosomal DNA, allowing for TF‐mediated activation of repressed chromatin. Moreover, there is evidence suggesting that certain TFs can repress actively expressed genes either directly through interactions with accessible regulatory elements or indirectly through mechanisms that impact the expression, activity, or localization of other regulatory factors. Recent evidence suggests that during reprogramming, the reprogramming TFs initiate opening of chromatin regions rich in somatic TF motifs that are inaccessible in the initial and final cellular states. It is postulated that analogous to a sponge, these transiently accessible regions “soak up” somatic TFs, hence lowering the initial barriers to cell fate changes. This indirect TF‐mediated gene regulation event, which is aptly named the “sponge effect,” may play an essential role in the silencing of the somatic transcriptional network during different cellular conversions.

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Reviewer's Responses to Questions
Is the topic timely and appropriate for the research community? Reviewer #1: I found this perspective article by Larcombe and colleagues well-presented and rather interesting, particularly their hypothesis of pioneer transcription factors enabling the sequestration of somatic transcription factors to enable somatic cell reprogramming. I only have some minor comments: 1-In the Abstract, I am not sure what is meant by direct, indirect, and secondary. 2-In page 4 line 54, I think that somatic cell nuclear transfer is at least as shocking conceptually as somatic cell reprogramming and transdifferentiation by exogenous factors. 3-NFKB is written wrong in multiple places. 4-I would suggest that the authors elaborate more on the previous hypothesis of pioneer transcription factors in somatic cell reprogramming (the 2 Cell papers by Soufi and colleagues). 5-I would also suggest that the authors discuss clearer that the Yamanaka factors can be activators and repressors, and in particular the role of c-MYC in repressing the somatic and pluripotent programs through interaction with NCOR/SMRT co-repressors (Nature Cell Biology 2018 by Zhuang and colleagues). 6-Do the authors think that the sponge effect versus the chromatin remodeling effect of the pioneer transcription factors might be different in mouse and human somatic cell reprogramming?
Reviewer #3: The review manuscript is generally well constructed and provides a thoughtful overview and discussion of the new mechanistic model to address a major gap in the cell reprogramming field; how to extinct pre-existing gene regulatory programs. Overall, this review is valuable scholarly literature, providing insights into cell fate changes in cell reprogramming, development, and tumorigenesis. Some areas need better clarification, as I will outline below.
* What the "sponge effect" represents was not explained in the Abstract and Introduction, even though it is the key mechanism to be discussed in the review. I had no idea about the concept until reading section 4. This needs to be explained earlier. * It is unclear whether sponge-like regions only function in redistributing somatic TFs in chromatin or also function as cis-regulatory regions for activating target genes during cellular reprogramming.
--Dr Andrew Hufton, Editor Advanced Genetics We would like to thank both reviewers for their time and valuable input. We have now addressed all of their comments and we believe that the manuscript is now of greater quality. Please find the response to all the points raised by the reviewers below: Reviewer #1: I found this perspective article by Larcombe and colleagues wellpresented and rather interesting, particularly their hypothesis of pioneer transcription factors enabling the sequestration of somatic transcription factors to enable somatic cell reprogramming. I only have some minor comments: 1. In the Abstract, I am not sure what is meant by direct, indirect, and secondary.
Thank you for bringing this to our attention. We realise that this isn't clear enough for an introductory statement and have clarified as follows:

ABSTRACT:
TFs are the master regulators of cellular identity, capable of driving cell fate transitions including differentiations, reprogramming and transdifferentiations. Pioneer TFs recognize partial motifs exposed on nucleosomal DNA, allowing for TF-mediated activation of repressed chromatin. Moreover, there is evidence suggesting that certain TFs can repress actively expressed genes either directly through interactions with accessible regulatory elements or indirectly through mechanisms that impact the expression, activity or localization of other regulatory factors. Recent evidence suggests that during reprogramming, the reprogramming TFs initiate opening of chromatin regions rich in somatic TF motifs that are inaccessible in the initial and final cellular states. We postulate that analogous to a sponge, these transiently accessible regions "soak up" somatic TFs, hence lowering the initial barriers to cell fate changes. This indirect TF-mediated event, which we've aptly named the "sponge effect", may play an essential role in the silencing of the somatic transcriptional network.
2. In page 4 line 54, I think that somatic cell nuclear transfer is at least as shocking conceptually as somatic cell reprogramming and transdifferentiation by exogenous factors.
We thank the reviewer for pointing this out and we agree that nuclear transfer experiments pioneered this field. We have now reworded the entire paragraph to acknowledge Robert Davis's TF-guided reprogramming and the prior discoveries made by John Gurdon's work.
While reprogramming into iPSCs is arguably the most prominent in vitro cellular conversion due to the unprecedented therapeutic potential of iPSCs, numerous other forced inter-cell conversions have been described. Pioneering work by Sir John Gurdon in 1962 demonstrated the potential of nuclear transfer as a means of changing cell identity and cloning. (Gurdon, 1962) In 1987, Davis and colleagues made another major scientific discovery in the field of cellular reprogramming demonstrating that fibroblasts can be converted into myoblasts by forcing the expression of the TF MYOD. 3. NFKB is written wrong in multiple places.
We are not exactly sure what is incorrect here. As far as we can see, NF-ⲕB is the correct written form for this transcriptional complex (as originally defined in 1986 by Ranjan Sen & David Baltimore's back-to-back Cell papers , 2006). We will alter the manuscript to initially define NF-ⲕB as nuclear factor-kB, however, we are happy to change this to what the editor believes is appropriate.
For example, uncontrolled activation of nuclear factor-kB (NF-ⲕB) blocks apoptosis, promotes cell cycle progression and limits innate and adaptive immune surveillance in several cancer cell types.
4. I would suggest that the authors elaborate more on the previous hypothesis of pioneer transcription factors in somatic cell reprogramming (the 2 Cell papers by Soufi and colleagues).
We appreciate the reviewers' input and agree that we should elaborate on Soufi et al. 's key findings regarding pioneer TFs and their activities in early reprogramming. Please see the end of the following paragraph in comment 5 as both comments are addressed in the same body of text.
5. I would also suggest that the authors discuss clearer that the Yamanaka factors can be activators and repressors, and in particular the role of c-MYC in repressing the somatic and pluripotent programs through interaction with NCOR/SMRT co-repressors (Nature Cell Biology 2018 by Zhuang and colleagues).
We agree with the reviewer that adding insight into the known roles of C-MYC during somatic cell exit and pluripotency acquisition prior to shifting focus improves the overall flow. We thank the reviewer for this suggestion and have adjusted the manuscript as outlined below.
To determine the molecular mechanisms underlying iPSC formation, several extensive epigenetic analyses have been conducted. (Chronis et al., 2017;Hussein et al., 2014;Knaupp et al., 2017a;Li et al., 2017a;Polo et al., 2012;Schwarz et al., 2018) Through the integration of various cell identity determinants including chromatin accessibility, reprogramming factor occupancy, DNA methylation, histone modifications and gene expression, each of these studies generally agree on several of the molecular aspects that underlie this cell conversion. Specifically, they all recognize the stage-specific and cooperative binding of reprogramming TFs, the rapid silencing of the somatic transcriptional network and the gradual activation of the pluripotency network during MEF reprogramming. (Chronis et al., 2017;Knaupp et al., 2017b;Li et al., 2017b) Furthermore, it is generally accepted that C-MYC, which greatly enhances reprogramming kinetics and efficiency, functions via a different mechanism to OCT4, SOX2 and KLF4 and plays a secondary role during this process. (Soufi et al., 2012) It has been reported that CMYC is a major contributor to the first wave of epigenetic events in the initial stages of reprogramming, in part through the recruitment of the NCoR/SMRT-HDAC3 complex that decommissions the somatic cell program.  (Chronis et al., 2017;Knaupp et al., 2017b;Li et al., 2017b) A major point of debate remains regarding how OCT4, SOX2 and KLF4, which seem to generally act as transcriptional activators, silence the somatic transcriptional network. This has led to the proposal of different mechanistic models for the TF-mediated loss of initial cellular identity (Figure 3).(J. Chen et al., 2016;X. Chen et al., 2008;Sridharan et al., 2009) Several studies have suggested that the reprogramming factors act as pioneer TFs at the start of reprogramming, predominantly targeting closed chromatin. (Roberts et al., 2021;Soufi et al., 2012Soufi et al., , 2015 One of the first studies to suggest that OCT4, SOX2 and KLF4 The degree to which these "sponge regions" are involved in the silencing of the somatic gene regulatory network during iPSC formation and whether it extents to other cellular conversions warrants further investigation. Reviewer #3: The review manuscript is generally well constructed and provides a thoughtful overview and discussion of the new mechanistic model to address a major gap in the cell reprogramming field; how to extinct pre-existing gene regulatory programs. Overall, this review is valuable scholarly literature, providing insights into cell fate changes in cell reprogramming, development, and tumorigenesis. Some areas need better clarification, as I will outline below.
1. What the "sponge effect" represents was not explained in the Abstract and Introduction, even though it is the key mechanism to be discussed in the review. I had no idea about the concept until reading section 4. This needs to be explained earlier.
We greatly appreciate the reviewer's feedback and have modified the abstract and the introduction to reiterate the sponge effect concept. We hope that this clarification of the sponge effect model better prepares the reader for the discussion in section 4.

Abstract:
TFs are the master regulators of cellular identity, capable of driving cell fate transitions including differentiations, reprogramming and transdifferentiations. Pioneer TFs recognize partial motifs exposed on nucleosomal DNA, allowing for TF-mediated activation of repressed chromatin. Moreover, there is evidence suggesting that certain TFs can repress actively expressed genes either directly through interactions with accessible regulatory elements or indirectly through mechanisms that impact the expression, activity or localization of other regulatory factors. Recent evidence suggests that during reprogramming, the reprogramming TFs initiate opening of chromatin regions rich in somatic TF motifs that are inaccessible in the initial and final cellular states. We postulate that analogous to a sponge, these transiently accessible regions "soak up" somatic TFs, hence lowering the initial barriers to cell fate changes. This indirect TF-mediated event, which we've aptly named the "sponge effect", may play an essential role in the silencing of the somatic transcriptional network.

Introduction:
In this perspective, we discuss the nature of pioneer TFs, the proposed mechanistic models behind direct and indirect TF-mediated cellular identity conversions and how these mechanistic aspects could be exploited to further enhance in vitro cell fate changes. Furthermore, we discuss how TF-mediated decondensation of motif-rich "sponge" chromatin can directly sequester TFs away from gene regulatory elements (REs) to expedite the extinction of established TF networks. We theorise that through this process, which we coined the "sponge effect", cell fate altering TFs initiate redistribution of TFs away from active REs to promote silencing of the initial cell identity network.

It is unclear whether sponge-like regions only function in redistributing somatic
TFs in chromatin or also function as cis-regulatory regions for activating target genes during cellular reprogramming.
We agree with the reviewer that one of the key questions regarding the sponge regions is whether they also (at least to some extent) act as regulatory elements. This in turn would mean that TF relocation to these sites has additional functional consequences apart from silencing of the initial cell identity network (e.g. activation of transiently expressed genes).
Indeed, our analysis of MEF reprogramming (Cell Stem Cell 2017) revealed that opening of these transiently accessible regions and expression of the "closest" genes correlate positively at the later stages of reprogramming. However, at the beginning of reprogramming, transient opening of these OCT4/SOX2 target sites does not correlate with transcriptional activation of the closest genes but with a decrease in expression of MEF identity genes and closure of MEF regulatory elements. This suggests that many of these transiently accessible chromatin regions indeed function as cis-regulatory elements towards the end of reprogramming when the cells transition towards pluripotency. However, the majority of these transiently accessible regions and OCT4/SOX2 target sites do not seem to act as cis-regulatory elements at the beginning of reprogramming or during the loss of somatic cell identity. Nevertheless, it is possible that these regions (at least some) act as distal regulatory elements as we acknowledge that the closest gene may not necessarily be the gene that is regulated. In order to address this interesting point raised by the reviewer, we have now added the following sentences for further clarification of the proposed sponge effect.
However, the majority of OCT4 and SOX2 binding events observed were to regions inaccessible in MEFs and iPSCs. (Knaupp et al., 2017a) Notably, transient opening of these chromatin regions does not generally correlate with transcriptional activation of the closest genes. Instead, opening of these regions coincides with silencing of the MEF identity network. This suggests that the majority of OCT4 and SOX2 target sites at the start of reprogramming are not cis-REs and most likely have different functions. Whether some of these transient regions act as distal REs remains largely unknown. Interestingly, these transiently accessible chromatin regions are not only enriched with the motifs of OCT4, SOX2 and KLF4 but also various somatic TF motifs including members of the AP-1, TEAD, RUNX and ETS TF families. (Knaupp et al., 2017a;Li et al., 2017a) Therefore, we proposed that OCT4, SOX2 and KLF4 engage with closed chromatin regions to open them transiently. This in turn initiates the redistribution of somatic factors to starve fibroblast identity genes of TFs, indirectly forcing the extinction of the somatic transcriptional network ( Figure 3C).
Editor: When revising your manuscript, please aim for concise wording wherever possible. In agreement with the reviewers, I felt that this Perspective was overall already quite well-written, but I do think there are some places where you may be able to streamline the wording. Please also keep an eye out for long paragraphs and consider breaking them into shorter paragraphs. I think the Introduction, for example, would be a good place where this may be helpful.
We would like to thank you for accepting our manuscript for publication in Advanced Genetics. We have addressed all reviewer comments and made several modifications to the original manuscript -highlighted in attached PDF. We have also broken the introduction into smaller paragraphs. We hope you find these changes satisfactory. Please see a summary of minor edits below: Blue highlights indicate where a paragraph was split into two.
The following sentence in the introduction: ○ TFs usually engage other regulatory proteins including other TFs, histone modifiers and chromatin remodelers to exert their functions. Was changed to: ○ To exert their function, TFs usually engage other regulatory proteins, including other TFs, histones modifiers and chromatin remodelers.
Insight into the underlying fundamental mechanisms through which TFs mediate changes in cell identity is therefore key to our understanding of human development and tumorigenesis. This basic knowledge fundamental information is of major relevance……